DESCRIPTION (provided by applica.nt): Human cytomegalovirus (HCMV) is a herpesvirus of considerable clinical importance and for which there exists a strong need for safe and effective antivirals. Although cleavage and packaging presents an attractive target for novel antivirals and several families of cleavage/packaging inhibitors have been discovered, our basic understanding of herpesvirus cleavage and packaging, and how these compounds act, remains scant. The parent grant represents an undertaking to identify the cis elements that are recognized during the cleavage and packaging process, the proteins that carry out cleavage and packaging, and the mechanism by which this concerted process occurs. In the parent proposal, experiments are described to express HCMV cleavage/packaging proteins and to characterize their biochemical functions in vitro. Because construction of complementing cell lines able to support replication of HCMV mutants in cleavage/packaging genes has thusfar been unsucessful, genetic approaches involving construction of viral mutants were not included in the parent grant. The advent of bacmid-cloned HCMV genomes facilitates rapid methods for recombinant virus construction that provide an alternative to conventional genetic analysis using complementing cell lines. Bacmid-cloned genomes with lethal deletions of essential viral genes can be created in E. coli and complementing genes can be restored to the mutant genomes in cis using highly rapid and reliable genetic tools such as Tn7 transposition, which we recently used to complement an HCMV ie2 mutant (Hahn et al., J. Virol. Meth., in press). Thus, a high throughput system is now available to evaluate the effects of small targeted mutations within cleavage/packaging genes in terms of growth/no-growth of the mutant viruses. This in itself will provide information critical to defining functional domains within these proteins. Moreover, if a sufficient number of small mutations are constructed, some will undoubtedly exhibit partial growth defects. Characterization of the phenotypes associated with these growth defects can proceed without complementing cells and should provide valuable clues to the functional domains within these proteins. Finally, identidication of second-site mutations that restore efficient growth may provide genetic evidence for functional interations between cleavage/packaging proteins.